Many prokaryotic and eukaryotic genes have been expressed at high levels in prokaryotes such as Escherichia coli. The general approach has been to use a multicopy cloning vector with a strong promoter and an efficient ribosome binding site for the transcription and translation of the cloned gene (Masui, Y., Coleman, J. and Inouye, M. (1983) in Experimental Manipulation of Gene Expression, ed. Inouye, M. (Academic, New York), pp. 15-32; Crowl, R., Seamans, C., Lomedico, P. and McAndrew, S. (1985) Gene 38:31-38). However, the level of gene expression with these vectors varies widely for different eukaryotic genes. Low-level expression has been attributed to protein degradation by E. coli proteases (Emerick, A. W., Bertolani, B. L., BenBassat, A., White, T. J. and Konrad, M. W. (1984) Bio/Technology 2:165-168) or to inefficient translation initiation of mRNAs containing heterologous gene sequences (Ray, P. N. and Pearson, M. L. (1974) J. Mol. Biol. 85:163-175; Ray, P. N. and Pearson, M. L. (1975) Nature (London) 253, 647-650; Kelley, R. L. and Yanofsky, C. (1982) Proc. Natl. Acad. Sci. USA 79:3120-3124; Nagai, K. and Thogersen, H. C. (1984) Nature (London) 309, 810-812; Varadarajan, R., Szabo, A. and Boxer, S. G. (1985) Proc. Natl. Acad. Sci. USA 82:5681-5684). Several studies suggested that the efficiency of translation initiation depends on the degree of complementarity between the Shine-Dalgarno (SD) sequence and the 16S rRNA, the distance between the SD sequence and the initiation codon, and the nucleotide sequence of this "window" region (Shine, J. and Dalgarno, L. (1975) Nature (London) 254, 34-38; Gold, L., Pribnow, D., Schneider, T., Shineding, S., Singer, B. S. and Stormo, G. (1981) Annu. Rev. Microbiol. 35: 365-403; Stromo, G. D., Schneider, T. D. and Gold, L. M. (1982) Nucleic Acids Res. 10:2971-2996; Kozak, M. (1983) Microbiol. Rev. 47:1-45; Hui, A., Hayflick, J., Dinkelspiel, K. and deBoer, H. A. (1984) EMBO J. 3:623-629; Shepard, M. G., Yelverton, E. and Goeddel, D. V. (1982) DNA 1:125-131; deBoer, H. A., Hui, A., Comstock, L. J., Wong, E. and Vasser, M. (1983) DNA 2:231-235; Whitehorn, E. A., Livak, K. J. and Petteway, S. R., Jr. (1985) Gene 36:375-379). There is evidence that the translational efficiency also depends on the sequence of the 5' untranslated region of the mRNA outside the SD sequence and the 5' end of the protein coding region (Stanssens, P., Remaut, E. and Fiers, W. (1985) Gene 36:211-223; Roberts, T. M., Kacich, R. and Ptashne, M. (1979) Proc. Natl. Acad. Sci. USA 76:760-764; Gold, L., Stormo, G. and Saunders, R. (1984) Proc. Natl. Acad. Sci. USA 81:7061-7065) and the 3' untranslated region of the mRNA.
To reconcile these observations, it has been proposed that translation is inhibited when local secondary structures form with regions containing the SD sequence and/or the AUG start codon such that the ribosomes cannot initiate translation (Gheysen, D., Iserentant, D., Derom, C. and Fiers, W. (1982) Gene 17:55-63; Iserentant, D. and Fiers, W. (1980) Gene 9:1-12; Schwartz, M., Roa, M. and Debarbouille, M. (1981) Proc. Natl. Acad. Sci. USA 78:2937-2941; Hall, M. N., Gabay, J., Debarbouille, M. and Schwartz, M. (1982) Nature (London) 295, 616-618; Das, A., Urbanowski, J., Weissbach, H., Nestor, J. and Yanofsky, C. (1983) Proc. Natl. Acad. Sci. USA 80:2879-2883; Berkhout, B. and van Duin, J. (1985) Nucleic Acids Res. 13:6955-6967). The formation of such secondary structures may explain failures to express methionyl bovine growth hormone (Met-bGH) with its native codons at high levels (George, H. J., L'Italien, J. J., Pilacinski, W. P., Glassman, D. L. and Krzyzek, R. A. (1985) DNA 4:273-281; Seeburg, P. H., Sias, S., Adelman, J., deBoer, H. A., Hayflick, J., Jhurani, P., Goeddel, D. V. and Heyneker, H. L. (1983) DNA2:37-45). To overcome this potential problem, Seeburg et al. have introduced several base changes into the 5' end of the bovine growth hormone (bGH) gene to create a sequence that is similar to the 5' end of the highly expressed human growth hormone (hGH) gene. Likewise, George et al. reported high-level expression (15% of total cell protein) after changing 13 codons in the 5' end of the bGH gene. These approaches are limited by the need to preserve the amino acid sequence of the protein. Polycistronic expression systems have been constructed to avoid the aforementioned limitations.
Features shared by polycistronic expression systems include a promoter to drive expression of the polycistronic mRNA, one or more ribosome binding sites, translation initiation sites for each cistron, and translation termination codons for each of the cistrons. The prior art teaches that expression levels of polypeptide products of interest are related to the strength of the promoter, the efficiency of ribosome binding site(s) on the polycistronic message, and the proper positioning of the translation initiation sites relative to the ribosome binding site(s) .
Even with the construction of polycistronic expression systems, the expression of both bovine growth hormone and its derivatives such as EK-BGH (Met-Phe-Pro-Leu-(Asp).sub.4 -Leu-BGH) remains problematic. Compounding the aforementioned problems is the structural instability of many expression vectors. Structural instability of recombinant DNA expression vectors results in DNA deletions and rearrangements that alter vector structure. This is a significant concern in large scale cultures grown to produce polypeptides encoded by these expression vectors. These vectors may be altered in a way that prevents expression of the encoded polypeptide. Thus, when the cultures are induced for expression of the polypeptide, a negative selective pressure toward a lack of polypeptide expression often results in an accumulation of the altered expression vectors.
In view of the above, regulatory agencies, such as the Food and Drug Administration, require full characterization of any recombinant DNA expression vectors that are utilized to produce polypeptide products of medicinal or veterinary utility. Evidence must be submitted to verify that the recombinant DNA expression vector is the same at the end of the fermentation as the expression vector from the original inoculum. Certification data includes structural and size analysis of the expression vector and verification of the nucleotide sequence that code for the desired product, and the regions flanking this coding sequence, especially flanking sequences that perform important functions, such as promoters.
Recombinant DNA vectors which utilize the Escherichia coli bacteriophage lambda pL promoter-operator region to enable transcription of an operably linked gene are often plagued by structural instability. When such vectors are examined at the end of the fermentation process, the structure of the vectors is often altered. The purpose of the present invention is to provide a stable expression vector while also providing regulatable transcription of the EK-BGH transcript.
The present invention provides an expression vector for production of EK-BGH which is stable, tightly regulated and achieve high levels of EK-BGH production. Thus, the present invention provides a significant advance in the area of production of EK-BGH and structurally related polypeptides.